Numerous methods are known to art which disclose the isolation of nucleic acids from sample material. To this end, methods for sample preparation are of high importance.
Particularly useful methods achieve this purpose by way of lysing the sample material and adsorbing nucleic acids to a solid phase (e.g., a silica matrix) in the presence of a chaotropic substance. By way of separating the select phase from the remaining lysate and subsequent desorption of the nucleic acid from the solid phase, the nucleic acid can be isolated efficiently.
During the adsorption process, the chaotropic substance effects removal of water molecules from the hydrate shell of dissolved nucleic acid molecules as well as from the surface of the solid phase, e.g., a silica matrix. As a result, a direct ionic interaction between the —Si—OH groups of the silica matrix and the phosphate-diester groups of the nucleic acid backbone becomes possible (Melzak, K. A., et al., J. Coll. Interf. Sci. 181 (1996) 635-644).
In the state of the art, the chaotropic substance is applied at very high concentrations in the range between 1 M to 6 M or even higher. Additives, e.g., other elements as boron, iron, phosphor, aluminum and the like, present in the silica matrix may influence the ability of the solid phase to bind to nucleic acids.
The described chaotropic effect is accompanied by an increase of the entropy. Thus, the equilibrium is shifted to the binding of the nucleic acid to the surface of the solid phase. As a prerequisite, however, the surface of the solid phase has to be in a neutral state. Especially for the surface of a silica material, the preferred pH range for adsorbing the nucleic acid is between pH 4 and pH 6.
The chaotropic effect can be enhanced by the addition of other dehydrating substances. For example, addition of an organic solvent, e.g., an alcohol, results in an improved adsorption of nucleic acids to glass surfaces. Alcohol concentrations usually are in the range between 30% and 60% [v/v]. In addition, alcohol appears to shift the selectivity towards binding nucleic acids, at the expense of other organic compounds.
Further, detergents are added at high concentrations (e.g., TRITON X-100, 20% [v/v]) to enhance lysis of the sample material. At the same time, detergents can positively influence the process of adsorption of nucleic acids to the solid phase.
A further advantage of chaotropic substances at high concentrations is an inhibition of nucleases which may be present in the lysate. This particular effect can be enhanced by adding reducing compounds such as dithiothreitol (DTT).
The state of the art has certain disadvantages. To achieve the desired adsorption of nucleic acids onto the solid phase compositions have to be formed which are very complex and which contain reagents—particularly one or more salts—at very high concentrations in order to achieve sufficient binding and selectivity. Depending on the complexity of the sample material before treatment, undesired constituents and particularly proteins have to be pretreated specifically. To this end, sample material is frequently digested with a proteinase, e.g., proteinase K. However, high concentrations of chaotropic substances inhibit proteinase activity. Although this shortcoming can be overcome by applying high quantities of proteolytic enzyme, this approach increases the costs of sample preparation since the protease needs to be of very high quality, that is to say, it must be free of nucleases. A further disadvantage is the frequent need of alcohol which is flammable and thus requiring safety precautions. Apart from safety concerns in the laboratory with regards to alcohol, this organic compound poses additional problems when being pipetted. The vapor pressure of alcohols such as ethanol or isopropanol is a particular technical problem with regards to automated handling of liquids.
It was therefore an object of the invention to provide alternative compositions for adsorbing a nucleic acid from a liquid phase to a solid phase. It was a particular object of the invention to provide compositions with organic additives which overcome at least some of the disadvantages of alcohols. A further particular object of the invention was to provide compositions which allow the adsorption of the nucleic acid to the solid phase in the presence of lower salt concentrations than in the state of the art.